Identification of Macrodomain Proteins as Novel O-Acetyl-ADP-ribose Deacetylases

Abstract

Sirtuins are a family of protein lysine deacetylases, which regulate gene silencing, metabolism, life span, and chromatin structure. Sirtuins utilize NAD ؉ to deacetylate proteins, yielding O-acetyl-ADP-ribose (OAADPr) as a reaction product. The macrodomain is a ubiquitous protein module known to bind ADP-ribose derivatives, which diverged through evolution to support many different protein functions and pathways. The observation that some sirtuins and macrodomains are physically linked as fusion proteins or genetically coupled through the same operon, provided a clue that their functions might be connected. Indeed, here we demonstrate that the product of the sirtuin reaction OAADPr is a substrate for several related macrodomain proteins: human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked MacroD-like protein from Staphylococcus aureus. In addition, we show that the cell extracts derived from MacroD-deficient Neurospora crassa strain exhibit a major reduction in the ability to hydrolyze OAADPr. Our data support a novel function of macrodomains as OAADPr deacetylases and potential in vivo regulators of cellular OAADPr produced by NAD ؉ -dependent deacetylation.Macrodomains are evolutionarily conserved structural domains found in proteins with diverse cellular functions (1, 2). Prior evidence suggested that macrodomains function as binding modules of NAD ϩ metabolites, including ADP-ribose/ poly(ADP-ribose) (3-7) and O-acetyl-ADP-ribose (OAADPr) 5 (8, 9). OAADPr is produced in reactions catalyzed by NAD ϩ -dependent protein/histone deacetylases (10, 11), which regulate gene silencing, metabolic enzymes, life span, and many other cellular processes (12)(13)(14). OAADPr has been implicated as a signaling molecule, modulating cellular processes affected by NAD ϩ -dependent protein/histone deacetylation (15-17). The binding of OAADPr and other NAD ϩ metabolites to macrodomains such as the histone variant macroH2A1.1 (1, 5, 8, 9) suggests a possible connection between metabolic regulation, gene activity, and chromatin structure. The mechanism by which cells regulate and utilize OAADPr is not well understood. In yeast, the NUDIX ADP-ribose pyrophosphatase Ysa1 modulates the cellular levels of both ADPr and OAADPr, converting each to AMP and the corresponding ribose-phosphate (17). Cells lacking ysa1 exhibit an increased resistance to oxidative insults and produce lower levels of endogenous reactive oxygen species. In vitro, the poly (ADP-ribose) glycohydrolase ARH3 was capable of removing the acetyl group from OAADPr (18), though the activity was orders of magnitude slower than that observed for the NUDIX family (19). In eukaryotic cell extracts at least two different cellular activities were shown to contribute to OAADPr deacetylation (19), but the identities of these factors remain unknown.Here we report a direct functional connection between sirtuins and a family of macrodomain proteins. We demonstrate that proteins belonging to this distinct branch of macrodomains are OAADPr deacetylases that...

Orphan Macrodomain Protein (Human C6orf130) Is an O-Acyl-ADP-ribose Deacylase

“…Tritium-labeled acetyl H3 peptide, H 2 N-KSTGGK( 3 H-acetyl)APRKQ-CONH 2 , was synthesized enzymatically from the 11-mer H3 peptide and purified as described previously (26). OAADPr, OPADPr, OBADPr, and O-3 Hacetyl-ADP-ribose ( 3 H-OAADPr) were synthesized enzymatically from ADPr and the acyl-peptides using yeast deacetylase HST2 and nicotinamidase from Salmonella enterica fused to maltose-binding protein (MBP-PncA) following procedures described previously (9,14). All other chemicals used were of the highest purity available commercially and were purchased from Sigma-Aldrich or Fisher Scientific.…”

“…Protein Expression and Purification for Enzymatic AssaysExpression and purification of yeast HST2 (14,27) and nicotinamidase from S. enterica fused to maltose-binding protein (28) were performed as described previously (9). Expression and purification of the His-tagged C6orf130 protein for enzymatic studies was achieved by transforming E. coli BL21(DE3) cells with the pDEST17 plasmid containing the C6orf130 gene insert and induction of mid-log phase cells (A 600 ϭ 0.7) with 0.1 mM isopropyl-1-thio-␤-D-galactopyranoside at 23°C.…”

“…It shares the least amino acid sequence similarity to the other MacroD-like proteins, among which human MacroD1 and MacroD2 are closely related and share more similarity to E. coli YmdB and the sirtuin-linked SAV0325 protein from S. aureus. The divergent amino acid sequence of C6orf130 makes it a distinct branch on the phylogenetic tree (9). C6orf130 transcription and expression are enriched in chronic lymphocytic leukemia cells and in B-cells of the chronic lymphocytic leukemia patients when effective graft-versus-leukemia responses are achieved after donor lymphocyte infusion (24).…”

“…Recently we have shown that a group of macrodomain proteins, i.e. MacroD-like proteins, including human MacroD1, human MacroD2, Escherichia coli YmdB, and the sirtuin-linked SAV0325 protein from Staphylococcus aureus, function as OAADPr deacetylases, catalyzing the deacetylation of OAADPr to form ADP-ribose and acetate (9). Mutagenesis and modeling of ADPr into the putative active site of MacroD1 suggested important roles for Asn-171, Asn-174, Asp-184, and His-188.…”

MACROD2 overexpression mediates estrogen independent growth and tamoxifen resistance in breast cancers

“…MACROD2 belongs to a family of genes containing a macro domain, an evolutionarily conserved protein motif (18), whose functional role until recently has been unclear. Studies have demonstrated that MACROD2 deacetylates O-acetyl-ADP ribose, a signaling molecule generated by the deacetylation of acetylated lysine residues in histones and other proteins (19). More recent work demonstrates that MACRO domain containing proteins are involved with mono-ADP ribosylation, and can regulate cell signaling pathways and modify proteins involved with gene transcription (20).…”